摘要
本文旨在克隆凋亡抑制因子Survivin基因,并在大肠杆菌中进行可溶性表达与初步纯化.采用RT-PCR法,扩增人凋亡抑制因子survivincDNA,并克隆入原核表达载体pMAL-p2X中,转化TB1大肠杆菌感受态细胞.经0.3mmol/LIPTG诱导2h后,收集菌体蛋白,进行SDS-PAGE、ELISA及Western印迹鉴定.实验获得凋亡抑制因子survivin编码区cDNA,以构建的原核表达载体pMAL-p2X-survivin转化菌株后,可表达凋亡抑制因子survivin和麦芽糖结合蛋白(MBP)的融合蛋白,相对分子质量(Mr)为58000.并成功利用FactorXa将融合蛋白裂解开.ELISA和Western印迹表明,融合蛋白能与抗凋亡抑制因子survivin单克隆抗体特异性结合.获得的凋亡抑制因子survivin全长cDNA可在大肠杆菌TB1中以MBP-survivin融合蛋白的形式表达,成功地将survivin目的蛋白和MBP蛋白分离,为深入研究survivin的结构和功能奠定了基础.
The mature peptide cDNA of human survivin, a apoptosis inhibitor, were cloned, and expressed in E. coli TB1 and purified. Using the isolated total RNA from human hepatocellular carcinoma cell line BEL7402 as a template , eDNA encoding the human survivin was amplified by RT-PCR. The PCR product was cloned into pGEM-T and sequenced. Then , gene encoding mature region of survivin was inserted into prokaryotic expression vector pMAL-p2X and protein was identified by SDS-PAGE and Western blot. The cloned gene sequence of transformed into competent E. coli TB1 to express via induction of IPTG. maltose binding protein-survivn fusion peptide was identical with that reported. SDS-PAGE and Western blot analysis indicated that the relative molecular mass （ Mr ） of the fusion protein was about 58 000, Fusion protein accounted for about 25% of total bacteria protein and could react specifically with anti-survivin antibody. eDNA encoding survivin has been cloned and expressed in E. coli TB1.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第3期200-204,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
北京市科委肿瘤分子生物学高技术实验室基金资助(编号953850500)~~
